Although in genetic engineering numerous polypeptide expression systems for prokaryotic or eukaryotic hosts are already known, there is a continuing need for novel systems which have advantages over the known systems.
Very widely used as hosts are the eukaryotic yeasts, e.g. Saccharomyces cerevisiae, for which different types of vectors exist. One type of vector are the integrating vectors which do not contain autonomously replicating sequences (ARS) and therefore, cannot be maintained extrachromosomally in the cell. This type of vector usually consists of yeast and bacterial DNA moieties. The vectors can integrate into the genome of a transformed yeast cell by homologous recombination. Despite the relatively high stability of the inserted DNA, these vectors have disadvantages as expression vectors for the production of recombinant proteins in yeast. The first disadvantage is the low transformation rate because a recombination event must occur in the transformed cell to produce a stable transformant. The second is that the copy number of the vector in the stably transformed cell is low.
Another type of vector is an extrachromosomally replicating vector. These vectors can be grouped into three categories:
The first group contains autonomously replicating sequences (ARS). These vectors are usually present in high copy numbers in the cell, however, they are often lost during cell division. PA1 The second group are so-called CEN vectors which contain a DNA sequence acting as a centromer during cell division. These vectors, though very stable, are present only in a few copies in the cells. PA1 The third group is derived from naturally occuring yeast plasmids, the two micron-like plasmids (for review see A. Hinnen and B. Meyhack, Current Topics in Microbiology and Immunology 96: 101-107, 1982, and B. Futcher, Yeast 4: 27-40, 1988). PA1 (a) culturing a yeast strain transformed with a symmetric two micron like hybrid vector containing an expression cassette consisting of a promoter functional in yeast, a signal sequence encoding a signal peptide functional in yeast, a structural gene coding for a yeast or non-yeast polypeptide, and optionally a terminator functional in yeast, PA1 (b) isolating said polypeptide
Several different two micron-like plasmids have been described. These are the two micron plasmid of S. cerevisiae, plasmids pSB1 and pSB2 from Zygosaccharomyces bailii, plasmid pSR2 from Z. rouxii, pSB3 from Z. bisporus, pSM1 from Z. fermentati, and pKD1 from Kluyveromyces drosophilarum. All seven plasmids are high copy number, double stranded circular DNA plasmids with remarkable similar structures: They all have two large exact inverted repeats diametrically opposed; they are found in two equimolar, isomeric forms; they all encode a FLP recombinase; they have an origin of replication immediately adjacent to one of the inverted repeats; all of the plasmids have at least three open reading frames. However, despite the structural and geometrical similarities, the plasmids share very little homology in their DNA sequence and in the amino acid sequences of the protein products of their genes (Futcher, op. cit.).
The naturally occuring two micron-like plasmids combine two essential features which are prerequisites for the use as starting vector in the construction of hybrid expression vectors for yeast: They are high copy number plasmids and are stably maintained in the cells.
The two micron plasmid of S. cerevisiae is found in the nucleus of almost all S. cerevisiae strains when isolated from the environment. The copy number of the two micron plasmid of S. cerevisiae is about 50 to 60 per cell. The rate of spontaneous plasmid loss is about 10.sup.-4 per cell per generation in rapidly growing haploid cells. Therefore it is not lost from S. cerevisiae populations in the laboratory except under extreme conditions.
The entire two micron plasmid of S. cerevisiae is 6318 base pairs in length. It has been fully sequenced (Hartley, J. L. and Donelson, J. E., Nature 286: 860-865, 1980). The plasmid contains two perfect 599 base pairs long inverted repeats, almost exactly diametrically opposed. The plasmid is found in two equimolar forms, A and B, which differ in the relative orientation of the two unique regions. The plasmid flips from one form the the other because of intramolecular recombination between the inverted repeats. The A and B forms of the plasmid are functionally equivalent.
The two micron plasmid contains four open reading frames: FLP, REP1, REP2 and D. Open reading frame FLP is a gene for a specific recombinase that causes the flipping of the A and B forms by recombining the inverted repeats. REP1 and REP2 are both required in cis or trans for plasmid stability. The D reading frame also encodes a gene involved in plasmid stability. The two micron plasmid also contains important cis-acting sites, e.g. an origin of replication from which the replication is initiated only once per S phase is located near the boundary of one of the inverted repeats. Another cis-acting element called STB or REP3 is involved in the stable inheritance of the plasmid to the daughter cells at mitosis. It seems to be the site of action of REP1 and REP2. Third, near the centre of each inverted repeat a small "FLP recognition target" (FRT) site is located at which the FLP recombinase acts.
The advantages of the naturally occuring two micron-like plasmids of yeast, i.e. stability and high copy number, are in general lost after the insertion of DNA. For example, the insertion of gene constructions containing either the PGK1, ADH1 or CYC1 promoter into the S. cerevisiae two micron plasmid resulted in a 4- to 10-fold decrease of the copy number within the cells in comparison with the two micron plasmid (B. E. Jordan et al., 15th Int. Conf. on Yeast Genetics and Molecular Biology, Abstr. No. S436).
A lot of attempts have been made in order to prepare two micron-like plasmid derived hybrid expression vectors which are more stable than the known ones. The attempts focussed for example on the insertion of foreign genes into such regions of the S. cerevisiae two micron plasmid which seemed not essential for the maintenance and copy number control of the plasmid in the cell. Other attempts focussed on the avoidance of bacterial DNA inserts in the two micron plasmid derived expression vectors. For example, in the PCT patent application WO88/08027 two micron plasmid derivatives are described which spontaneously loose the bacterial DNA sequences in yeast cells.
In the method used in said PCT application, bacterial DNA which is required for the propagation and multiplication of the two micron derived expression vectors in bacterial host cells, is inserted between two directly inverted FRT sites. A third FRT site is located on the plasmid in indirect orientation. After transformation of a yeast cell, the DNA located between the two FRT sites in direct orientation is deleted due to a homologous recombination event and a two micron derived plasmid without bacterial sequences arises in the yeast cell. A two micron plasmid derived vector which looses part of its DNA due to homologous recombination between two directly orientatd FRT sites is called a "disintegration vector".
It is an object of the present invention to provide futher stable yeast expression plasmids which are derived from a two micron plasmid.